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- PDB-6ee8: Mycobacterium tuberculosis RNAP promoter unwinding intermediate c... -

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Basic information

Entry
Database: PDB / ID: 6ee8
TitleMycobacterium tuberculosis RNAP promoter unwinding intermediate complex with RbpA/CarD and AP3 promoter
Components
  • (DNA-directed RNA polymerase subunit ...Polymerase) x 4
  • (RNA polymerase-binding ...) x 2
  • DNA (60-MER)
  • DNA (65-MER)
  • RNA polymerase sigma factor SigA
KeywordsTRANSCRIPTION/DNA / initiation / half bubble / open promoter complex / TRANSCRIPTION / intermediate / TRANSCRIPTION-DNA complex
Function / homologyRNA polymerase Rpb2, OB-fold / RNA polymerase-binding protein RbpA / DNA-directed RNA polymerase, insert domain superfamily / RNA polymerase, RBP11-like subunit / Winged helix-like DNA-binding domain superfamily / RPB6/omega subunit-like superfamily / CarD-like/TRCF domain superfamily / RNA polymerase sigma factor RpoD / DNA-directed RNA polymerase, beta subunit, external 1 domain / RNA polymerase Rpb2, domain 2 superfamily ...RNA polymerase Rpb2, OB-fold / RNA polymerase-binding protein RbpA / DNA-directed RNA polymerase, insert domain superfamily / RNA polymerase, RBP11-like subunit / Winged helix-like DNA-binding domain superfamily / RPB6/omega subunit-like superfamily / CarD-like/TRCF domain superfamily / RNA polymerase sigma factor RpoD / DNA-directed RNA polymerase, beta subunit, external 1 domain / RNA polymerase Rpb2, domain 2 superfamily / DNA-directed RNA polymerase, subunit 2 / RNA polymerase sigma-70 like domain / RNA polymerase sigma factor, region 2 / RNA polymerase sigma factor, region 3/4-like / RNA polymerase sigma factor RpoD, C-terminal / DNA-directed RNA polymerase, subunit beta-prime / DNA-directed RNA polymerase, subunit 2, hybrid-binding domain superfamily / RNA polymerase Rpb1, funnel domain superfamily / RNA polymerase Rpb1, domain 1 / Sigma-70 region 3 / RNA polymerase Rpb2, domain 3 / RNA polymerase beta subunit / RNA polymerase Rpb2, domain 2 / RNA polymerase Rpb2, domain 7 / Sigma-70, region 4 / Sigma-70 region 2 / Bacterial RNA polymerase, alpha chain C terminal domain / RbpA superfamily / CarD-like/TRCF domain / RNA polymerase Rpb3/Rpb11 dimerisation domain / RNA polymerase Rpb6 / RNA polymerase Rpb3/RpoA insert domain / RNA polymerase Rpb1, domain 2 / RNA polymerase Rpb2, domain 6 / Sigma-70 factor, region 1.2 / RNA polymerase subunit, RPB6/omega / DNA-directed RNA polymerase, alpha subunit / DNA-directed RNA polymerase, RpoA/D/Rpb3-type / RNA polymerase Rpb1, domain 1 / RNA polymerases beta chain signature. / Sigma-70 factors family signature 2. / Sigma-70 factors family signature 1. / RNA polymerase-binding protein / RNA polymerase beta subunit external 1 domain / RNA polymerase Rpb1, domain 4 / RNA polymerase Rpb1, domain 5 / RNA polymerase, alpha subunit / RNA polymerase sigma-70 / CarD-like/TRCF domain / DNA-directed RNA polymerase, omega subunit / RNA polymerase, subunit omega/K/RPB6 / RNA polymerase, N-terminal / DNA-directed RNA polymerase, insert domain / RNA polymerase Rpb1, domain 3 / RNA polymerase Rpb1, domain 5 / RNA polymerase Rpb1, domain 4 / RNA polymerase, alpha subunit, C-terminal / DNA-directed RNA polymerase beta subunit, bacterial-type / RNA polymerase sigma-70 region 1.2 / RNA polymerase Rpb2, domain 3 / RNA polymerase, beta subunit, protrusion / RNA polymerase Rpb1, domain 3 / RNA polymerase Rpb2, domain 2 / RNA polymerase Rpb2, domain 7 / RNA polymerase sigma-70 region 4 / RNA polymerase sigma-70 region 2 / RNA polymerase sigma-70 region 3 / RNA polymerase, beta subunit, conserved site / DNA-directed RNA polymerase, subunit 2, hybrid-binding domain / bacterial-type RNA polymerase core enzyme binding / transcription initiation from bacterial-type RNA polymerase promoter / sigma factor activity / ribonucleoside binding / DNA-directed 5'-3' RNA polymerase activity / DNA-directed RNA polymerase / pathogenesis / protein dimerization activity / transcription, DNA-templated / DNA-binding transcription factor activity / positive regulation of transcription, DNA-templated / DNA binding / cytoplasm / DNA-directed RNA polymerase subunit omega / DNA-directed RNA polymerase subunit beta' / DNA-directed RNA polymerase subunit alpha / RNA polymerase sigma factor SigA / RNA polymerase-binding protein RbpA / RNA polymerase-binding transcription factor CarD / DNA-directed RNA polymerase subunit beta
Function and homology information
Specimen sourceMycobacterium tuberculosis (bacteria)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / 3.92 Å resolution
AuthorsDarst, S.A. / Campbell, E.A. / Boyaci Selcuk, H. / Chen, J.
CitationJournal: Nature / Year: 2019
Title: Structures of an RNA polymerase promoter melting intermediate elucidate DNA unwinding.
Authors: Hande Boyaci / James Chen / Rolf Jansen / Seth A Darst / Elizabeth A Campbell
Validation Report
SummaryFull reportAbout validation report
DateDeposition: Aug 13, 2018 / Release: Nov 21, 2018
RevisionDateData content typeGroupCategoryItemProviderType
1.0Nov 21, 2018Structure modelrepositoryInitial release
1.1Jan 23, 2019Structure modelData collection / Database referencescitation / citation_author_citation.country / _citation.journal_abbrev / _citation.journal_id_ASTM / _citation.journal_id_CSD / _citation.journal_id_ISSN / _citation.pdbx_database_id_DOI / _citation.pdbx_database_id_PubMed / _citation.title / _citation.year
1.2Jan 30, 2019Structure modelData collection / Database referencescitation_citation.journal_volume / _citation.page_first / _citation.page_last

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Structure visualization

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Assembly

Deposited unit
A: DNA-directed RNA polymerase subunit alpha
B: DNA-directed RNA polymerase subunit alpha
C: DNA-directed RNA polymerase subunit beta
D: DNA-directed RNA polymerase subunit beta'
E: DNA-directed RNA polymerase subunit omega
F: RNA polymerase sigma factor SigA
J: RNA polymerase-binding protein RbpA
O: DNA (65-MER)
P: DNA (60-MER)
M: RNA polymerase-binding transcription factor CarD
hetero molecules


Theoretical massNumber of molelcules
Total (without water)505,63913
Polyers505,48410
Non-polymers1553
Water0
1


TypeNameSymmetry operationNumber
identity operation1_5551

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Components

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DNA-directed RNA polymerase subunit ... , 4 types, 5 molecules ABCDE

#1: Protein/peptide DNA-directed RNA polymerase subunit alpha / Polymerase / RNAP subunit alpha / RNA polymerase subunit alpha / Transcriptase subunit alpha


Mass: 37745.328 Da / Num. of mol.: 2 / Source: (gene. exp.) Mycobacterium tuberculosis (bacteria) / Gene: rpoA / Production host: Escherichia coli (E. coli) / References: UniProt: A5U8D3, DNA-directed RNA polymerase
#2: Protein/peptide DNA-directed RNA polymerase subunit beta / Polymerase / RNAP subunit beta / RNA polymerase subunit beta / Transcriptase subunit beta


Mass: 125390.875 Da / Num. of mol.: 1 / Source: (gene. exp.) Mycobacterium tuberculosis (bacteria) / Gene: rpoB, SAMEA2682864_01701 / Production host: Escherichia coli (E. coli) / References: UniProt: V9Z879, DNA-directed RNA polymerase
#3: Protein/peptide DNA-directed RNA polymerase subunit beta' / Polymerase / RNAP subunit beta' / RNA polymerase subunit beta' / Transcriptase subunit beta'


Mass: 148202.219 Da / Num. of mol.: 1 / Source: (gene. exp.) Mycobacterium tuberculosis (bacteria) / Gene: rpoC / Production host: Escherichia coli (E. coli) / References: UniProt: A5U053, DNA-directed RNA polymerase
#4: Protein/peptide DNA-directed RNA polymerase subunit omega / Polymerase / RNAP omega subunit / RNA polymerase omega subunit / Transcriptase subunit omega


Mass: 11776.996 Da / Num. of mol.: 1 / Source: (gene. exp.) Mycobacterium tuberculosis (bacteria)
Gene: rpoZ, ERS007672_03979, ERS007703_04032, ERS007720_04749, ERS027652_00548, ERS027654_02543, ERS027656_03959, ERS124361_02246
Production host: Escherichia coli (E. coli)
References: UniProt: A0A0T9N9K3, DNA-directed RNA polymerase

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Protein/peptide , 1 types, 1 molecules F

#5: Protein/peptide RNA polymerase sigma factor SigA / Sigma-A


Mass: 58169.477 Da / Num. of mol.: 1 / Source: (gene. exp.) Mycobacterium tuberculosis (bacteria) / Gene: sigA, mysA, rpoD, rpoV / Production host: Escherichia coli (E. coli) / References: UniProt: P9WGI0

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RNA polymerase-binding ... , 2 types, 2 molecules JM

#6: Protein/peptide RNA polymerase-binding protein RbpA


Mass: 12993.695 Da / Num. of mol.: 1 / Source: (gene. exp.) Mycobacterium tuberculosis (bacteria) / Gene: rbpA / Production host: Escherichia coli (E. coli) / References: UniProt: P9WHJ4
#9: Protein/peptide RNA polymerase-binding transcription factor CarD


Mass: 17933.361 Da / Num. of mol.: 1 / Source: (gene. exp.) Mycobacterium tuberculosis (bacteria) / Gene: carD / Production host: Escherichia coli (E. coli) / References: UniProt: P9WJG2

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DNA chain , 2 types, 2 molecules OP

#7: DNA chain DNA (65-MER)


Mass: 27907.809 Da / Num. of mol.: 1 / Source: (synth.) Mycobacterium tuberculosis (bacteria)
#8: DNA chain DNA (60-MER)


Mass: 27618.625 Da / Num. of mol.: 1 / Source: (synth.) Mycobacterium tuberculosis (bacteria)

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Non-polymers , 2 types, 3 molecules

#10: Chemical ChemComp-ZN / ZINC ION


Mass: 65.409 Da / Num. of mol.: 2 / Formula: Zn / Zinc
#11: Chemical ChemComp-MG / MAGNESIUM ION


Mass: 24.305 Da / Num. of mol.: 1 / Formula: Mg / Magnesium

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Experimental details

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Experiment

ExperimentMethod: ELECTRON MICROSCOPY
EM experimentAggregation state: PARTICLE / Reconstruction method: single particle reconstruction

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Sample preparation

ComponentName: Mycobacterium tuberculosis RNAP promoter unwinding intermediate
Type: COMPLEX / Entity ID: 1, 2, 3, 4, 5, 6, 7, 8, 9 / Source: RECOMBINANT
Source (natural)Organism: Mycobacterium tuberculosis (bacteria)
Source (recombinant)Organism: Escherichia coli (E. coli)
Buffer solutionpH: 8
SpecimenEmbedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
Specimen supportDetails: unspecified
VitrificationCryogen name: ETHANE

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Electron microscopy imaging

Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company
MicroscopyMicroscope model: FEI TITAN KRIOS
Electron gunElectron source: FIELD EMISSION GUN / Accelerating voltage: 300 / Illumination mode: FLOOD BEAM
Electron lensMode: BRIGHT FIELDBright-field microscopy
Image recordingElectron dose: 1.4 / Film or detector model: GATAN K2 SUMMIT (4k x 4k)

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Processing

SoftwareName: PHENIX / Version: 1.13_2998: / Classification: refinement
CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
3D reconstructionResolution: 3.92 / Resolution method: FSC 0.143 CUT-OFF / Number of particles: 140333 / Symmetry type: POINT
Refine LS restraints
Refine IDTypeDev idealNumber
ELECTRON MICROSCOPYf_bond_d0.00730543
ELECTRON MICROSCOPYf_angle_d0.94841907
ELECTRON MICROSCOPYf_dihedral_angle_d13.48918224
ELECTRON MICROSCOPYf_chiral_restr0.0554775
ELECTRON MICROSCOPYf_plane_restr0.0075070

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